Abstract Background In vitro cell culture models provide a reproducible, cost-effective assessment of the effects of exposure to particulate matter (PM) and its components. While much consideration is paid to the cells, how composition and complexity of culture media may influence PM studies is poorly understood. Objective To determine the effects of culture media composition on immortalised alveolar epithelial cell (AEC) responses to vanadium pentoxide (V2O5), a tracer of heavy fuel oil combustion associated with respiratory and cardiovascular effects. Methods AECs were exposed to 0 to 100 µM V2O5 for 24 h in five different media (DCCM-1, DMEM:F12 (1:1), Hybridoma (Sartorius), Hybridoma (ThermoFisher) or Plasmax (CancerTools.org)). Expression of metal-, oxidative stress-, and viral response-genes was measured by RT-qPCR, and inflammatory mediator release by ELISA. Composition of media and post-exposure AECs was measured by inductively coupled plasma-mass spectrometry. Results In DCCM-1, V2O5 elicited upregulation of metallothionein and antioxidant gene expression (MT1G: 120-fold, HMOX1: 13-fold, both P < 0.001), which was muted in other media. Media-dependent differences in IL-6 release were also observed. Conversely, V2O5-mediated inhibition of antiviral genes (IFIT1, ISG15: 0.07 to 0.10-fold, P < 0.05) was robust across media. V2O5 elicited a concentration-dependent increase in intracellular vanadium across all media, but in DCCM-1, which contained higher copper concentrations, this was accompanied by increased intracellular copper. Supplementation of DMEM:F12 with copper replicated the effects of V2O5 in DCCM-1. Conclusions V2O5 induces metallothionein and antioxidant responses in a copper-dependent manner. The mechanism of copper entry remains under investigation. These findings highlight the need for careful consideration and reporting of cell culture conditions.

Exposure to ambient airborne particulate matter (PM) has been associated with almost 9 million premature deaths per year worldwide. Ports are essential infrastructure for trade and transport, with over 80% of the world’s international trade volume travelling by sea, yet there is little understanding of the potential health effects of PM from ships. Here, we studied the elemental and toxicological characteristics of size-fractionated PM collected at different areas of a busy European mixed-use port. We found that unregulated ultrafine PM, a PM size fraction <100 nm in aerodynamic diameter, originating from a cruise ship source, was highly enriched compared to ultrafine PM from other sites in tracers of heavy fuel oil combustion (V and Ni), with Co identified as a novel tracer. In cell models of airway and alveolar epithelium, this cruise ship-associated ultrafine PM was markedly more pro-inflammatory than PM from other sites, while simultaneously reducing expression of a range of genes associated with the innate antiviral response. Investigation of the effects of the three metals found to be enriched in our cruise ship ultrafine PM showed that these effects could be reproduced by vanadium exposure, which reduced interferon signalling and increased permissiveness to viral infection, facilitating replication of the respiratory viruses rhinovirus-16 and SARS-CoV-2. Our findings suggest that, despite a series of regulations introduced over the last 20 years, cruise ship-associated emissions of PM from heavy fuel oil combustion may exert disproportionate effects on health compared to PM from other sources. In particular, the effects manifest themselves through induction of a response with characteristics of a poor viral infection prognosis. More broadly, current regulations, which neglect PM source- and composition-related variations, and ultrafine PM in general, may need strengthening to better mitigate health effects resulting from exposure to PM.

Abstract Background Exposure to airborne particulate matter (PM) has been associated with adverse effects on the respiratory system. Shipping-associated PM is an important contributor to airborne PM in port and coastal areas, but composition-specific effects are poorly understood. Objective To investigate the effects of shipping-associated PM on bronchial and alveolar epithelial cells. Methods Coarse, fine and ultrafine PM (UFPM) was collected near a cruise shipping terminal at the Port of Southampton. Composition was characterised by inductively coupled plasma-mass spectrometry. 16HBE14o- bronchial epithelial cells exposed to 100 µg/mL PM for 24 h were analysed transcriptomically by RNA-seq. Effects of metals enriched in cruise ship UFPM were studied in alveolar epithelial cells (AEC). Gene and protein expression were measured by RT-qPCR, and Western blotting, and inflammatory mediator release by ELISA. Effects on viral replication were studied by viral copy number RT-qPCR in human primary bronchial epithelial cells infected with rhinovirus-16 (RV-16), and focus-forming assay in VeroE6 cells infected with SARS-CoV-2. Results Cruise shipping-associated UFPM was enriched in vanadium, nickel and cobalt, inducing inflammatory mediator release in 16HBE14o-. RNA-Seq revealed significant UFPM-induced downregulation of interferon signalling pathways. In AEC this was recapitulated by vanadium, inducing downregulation of antiviral genes (ISG15: 0.22-fold, IFIT1: 0.10-fold, P ≤ 0.05). Similar vanadium-mediated inhibition was observed following stimulation by interferon-β- and poly(I:C). Replication of RV16 and SARS-CoV-2 was increased by vanadium exposure. Conclusions Shipping-associated UFPM impairs antiviral signalling and drives a pro-inflammatory response, effects recapitulated by vanadium. Further research is needed to understand the implications of these findings in vivo.

Exposure to air pollution has been associated with up to 9 million premature deaths per year worldwide, with the respiratory system a key site for its effects. Air pollution exposure is a well-established risk factor for the development and exacerbation of airways diseases and lung cancer, however relatively little is known regarding the risks associated with air pollution interacting with areas of gas exchange - the alveoli and pulmonary interstitium. In recent years, evidence has emerged identifying a role in the development and progression of sub-clinical interstitial lung abnormalities as well as progression and risk of exacerbation of fibrotic interstitial lung diseases. This review outlines the epidemiologic evidence that air pollution perturbs alveolar health. It considers the different components of ambient air pollution, how penetration to the alveoli is determined by particle size and whether the response to alveolar exposure may be modulated by personal susceptibility factors. We discuss potential acute and chronic pathogenic mechanisms of injury upon the pulmonary interstitium and how these may contribute to the development and/or progression of interstitial processes. Finally, we explore current knowledge gaps and the potential for air pollution interventions in vulnerable individuals to support alveolar homeostasis and so prevent disease development and/or progression.

INTRODUCTION:Following the INPULSIS and ASCEND studies, leading to the first two approved antifibrotic therapies for patients with IPF, ongoing investigations are firmly exploring novel agents for a targeted effective and better tolerated therapy able to improve the natural history of the disease.AREAS COVERED:This review aims to analyze recent advances in pharmacological research of IPF, discussing the currently available treatments and the novel drugs under investigation in phase 3 trials, with particular emphasis on BI 1015550 and inhaled treprostinil. The literature search utilized Medline and Clinicaltrials.org databases. Critical aspects of clinical trial design in IPF are discussed in light of recently completed phase III studies.EXPERT OPINION:While randomized clinical trials in IPF are currently underway, future objectives should explore potential synergistic benefits when combining novel molecules with the existing therapies and identify more specific molecular targets. Moreover, refining the study design represent another crucial goal. The aim of the pharmacological research will be not only stabilizing but also potentially reversing the fibrotic changes in IPF.

INTRODUCTION:Currently approved drug treatments for idiopathic pulmonary fibrosis (IPF), pirfenidone and nintedanib, have been shown to slow lung function decline and improve clinical outcomes. Since significant advances in the understanding of pathogenetic mechanisms in IPF, novel potential agents are being tested to identify new targeted and better tolerated therapeutic strategies.AREAS COVERED:This review describes the evidence from IPF phase II and III clinical trials that have been completed or are ongoing in recent years. The literature search was performed using Medline and Clinicaltrials.org databases. Particular attention is paid to the new inhibitor of phosphodiesterase 4B (BI 1015550), being studied in a more advanced research phase. Some emerging critical issues of the pharmacological research are highlighted considering the recent outstanding failures of several phase III trials.EXPERT OPINION:An exponential number of randomized clinical trials are underway testing promising new molecules to increase treatment choices for patients with IPF and improve patients' quality of life. The next goals should aim at a deeper understanding of the pathogenic pathways of the disease with the challenging goal of being able not only to stabilize but also to reverse the ongoing fibrotic process in patients with IPF.

Novel approach of asthma includes personalised therapy involving specific immune pathways. We describe here a case of T2-high asthma in a 66-year-old woman treated with maximal inhaled therapy and inappropriate usage of oral corticosteroids showing poor symptoms control. Both anti-IgE and (omalizumab) and anti-interleukin (IL)-5 (mepolizumab) monoclonal antibodies treatments were prescribed without significant benefit. Add-on subcutaneous dupilumab, a monoclonal antibody directed against the IL-4 receptor subunit alpha, inhibiting signalling from both IL-4 and IL-13, proved to be an effective and safe medication to obtain rapid asthma control. Considering the previous lack of response to both anti-IgE and anti-eosinophilic strategies, we hypothesise that dupilumab upstream activity could exert different and more relevant effects than the simple inhibition of the two single downstream pathways. The current case highlights the need for a deeper analysis of biomolecular interactions in the framework of different asthma endotypes, to identify peculiar profiles associated with specific treatment responses.

Purpose of review In chronic pulmonary sarcoidosis, the transition from the inflammatory to the fibrotic stage of the lungs occurs in about 10–20% of cases, eventually causing end-stage fibrotic disease. To date, pathogenetic mechanisms and clinical management remain challenging; thus, we highlight the recent evidence in pulmonary fibrotic processes, clinical signs for an early detection and the potential role of the current investigated antifibrotic agents and promising targeted therapies. Recent findings Recent findings of relevant key cellular pathways can be considered as a glimmer of light in the complexity of sarcoidosis. In some patients, granulomas persist and serve as a nidus for fibrosis growth, sustained by several fibrosis-stimulating cytokines. Preclinical studies have detected profibrotic, antifibrotic and pleiotropic T cells as promoters of fibrosis. Epigenetics, genetics and transcriptomics research can lead to new target therapies. Antifibrotic drug nintedanib has shown a positive effect on non-idiopathic pulmonary fibrosis fibrotic lung diseases including fibrotic sarcoidosis; other antifibrotic drugs are under investigation. Summary Pulmonary fibrosis strongly impacts the outcome of sarcoidosis, and a better understanding of the underlying pathogenic mechanisms can facilitate the development of novel treatments, improving clinical care and life expectancy of these patients. The greatest challenge is to investigate effective antifibrotic therapies once fibrosis develops. The role of these findings in fibrotic sarcoidosis can be translated into other interstitial lung diseases characterized by the coexistence of inflammatory and fibrotic processes.

Lung ultrasound (LUS) imaging is playing an important role in the current pandemic, allowing the evaluation of patients affected by COVID-19 pneumonia. However, LUS is limited to the visual inspection of ultrasound data, which negatively affects the reproducibility and reliability of the findings. For these reasons, we were the first to propose a standardized imaging protocol and a scoring system, from which we developed the first artificial intelligence (AI) models able to evaluate LUS videos. Furthermore, we demonstrated prognostic value of our approach and its utility for patients' stratification. In this study, we report on the level of agreement between AI and LUS clinical experts (MD) on LUS data acquired from both COVID-19 patients and post-COVID-19 patients.

Lung ultrasound (LUS) is currently utilized worldwide to assess COVID-19 patients. However, imaging protocols are often defined arbitrarily, and studies on post-COVID-19 are lacking. In this work, we report on the capabilities of standardized LUS to monitor and stratify COVID-19 and post-COVID-19 patients. A validated and standardized imaging protocol based on 14 scanning-areas and a 4-level scoring system were utilized to collect and analyze data from 220 patients, 100 COVID-19 positive, and 120 post-COVID-19. Next, the capability of five imaging protocols (based on 4, 8, 10, 12, and 14 scanning-areas) to intercept the most significant LUS findings was compared. Moreover, a longitudinal-study was conducted aiming at investigating the possibility to simplify the protocol during follow-up. Results on the agreement between AI-models and LUS experts with respect to LUS data evaluation are also reported. In conclusion, a 12-areas protocol emerges as the optimal trade-off between a time-efficient and an accurate LUS examination. However, it appears not to be possible to reduce further the number of scanning-areas during follow-up. Finally, COVID-19 and post-COVID-19 data seem to show differences capable to confuse AI models that were not trained on post-COVID-19 data, supporting the hypothesis of the existence of LUS patterns specific to post-COVID-19.

To the Editors, Pulmonary involvement in Crohn’s disease (CD) remains poorly characterized due to its uncommon detection and a wide spectrum of manifestations. Large airway stenosis, bronchiolitis, bronchiectasis, organizing pneumonia, and alveolitis have been described, but acute respiratory distress syndrome (ARDS) has been reported only in a few cases.1 An association between surgery for inflammatory bowel disease and development of symptomatic lung disease has also been noted in susceptible patients.2 A 16-year-old female has been in follow-up at the Crohn’s disease outpatient clinic of our hospital (Fondazione Policlinico Universitario A. Gemelli IRCCS) since 2015, suffering from a stage A1B2L3 CD, according to Montreal classification. She was on treatment with infliximab (IFX). In April 2020, a steroid regimen with oral methylprednisolone (40mg/day for 10 days) was introduced because of the worsening of the symptoms. The diagnostic workup showed a worsening of ascending colon and terminal ileum stenotic lesions. Despite...

Lung ultrasound (LUS) has been reported as a useful tool to intercept lung peripheral changes (LPC) in COVID-19 pneumonia. Sixteen confirmed COVID-19 pneumonia patients underwent LUS using a standard sequence of scans in 14 landmarks. A score ranging from 0 to 3, according to Soldati's proposal, was reported for each landmark. High-resolution CT-scan of the chest (HRCT) was performed within 48 h prior to or after LUS. For each corresponding HRCT area, was reported a score (0 normal peripheral lung, 1 minimal LPC, 2 peripheral ground glass opacities (GGOs), 3 peripheral lung consolidations with or without GGOs) LUS showed sensitivity 92.1%, specificity 90%, PPV 96.8% to intercept LPC on HRCT (scores ≠ 0). Higher LUS scores (2–3), corresponding to worst changes, showed sensitivity 70.1%, specificity 84%, PPV 78.1% to intercept higher HTCT scores (2–3). The overall score, for both LUS and HRCT, over 14 landmarks, showed no significant differences (paired t-test p = 0.055). An overall score ≥24 was reported in five cases by LUS and 6 cases by HRCT. No significant differences also for patients either with more than three landmarks with score 3 or with 8 landmarks out of 14 with score 2–3 (p = 0.16). LUS showed good sensitivities and specificities compared to HRCT.

OBJECTIVES:Worldwide, lung ultrasound (LUS) was utilized to assess coronavirus disease 2019 (COVID-19) patients. Often, imaging protocols were however defined arbitrarily and not following an evidence-based approach. Moreover, extensive studies on LUS in post-COVID-19 patients are currently lacking. This study analyses the impact of different LUS imaging protocols on the evaluation of COVID-19 and post-COVID-19 LUS data.METHODS:LUS data from 220 patients were collected, 100 COVID-19 positive and 120 post-COVID-19. A validated and standardized imaging protocol based on 14 scanning areas and a 4-level scoring system was implemented. We utilized this dataset to compare the capability of 5 imaging protocols, respectively based on 4, 8, 10, 12, and 14 scanning areas, to intercept the most important LUS findings. This to evaluate the optimal trade-off between a time-efficient imaging protocol and an accurate LUS examination. We also performed a longitudinal study, aimed at investigating how to eventually simplify the protocol during follow-up. Additionally, we present results on the agreement between AI models and LUS experts with respect to LUS data evaluation.RESULTS:A 12-areas protocol emerges as the optimal trade-off, for both COVID-19 and post-COVID-19 patients. For what concerns follow-up studies, it appears not to be possible to reduce the number of scanning areas. Finally, COVID-19 and post-COVID-19 LUS data seem to show differences capable to confuse AI models that were not trained on post-COVID-19 data, supporting the hypothesis of the existence of LUS patterns specific to post-COVID-19 patients.CONCLUSIONS:A 12-areas acquisition protocol is recommended for both COVID-19 and post-COVID-19 patients, also during follow-up.

Introduction: Heart failure (HF) affects approximately 2% of the population worldwide, remaining a major cause of hospitalization and mortality despite innovative therapeutic approaches introduced in the past few decades. Type 2 diabetes mellitus (T2DM) contributes significantly to end-organ damage and HF-related complications and is associated with worse clinical status and increased all-cause and cardiovascular mortality in patients with HF with reduced (HFrEF) or with preserved ejection fraction (HFpEF), compared to HF patients without T2DM. Recently, a novel class of antidiabetic drugs has been introduced: sodium glucose co-trasport-2 inhibitors (SGLT2i). Initially designed for patients with T2DM to reduce kidney blood glucose resorption, SGLT2i rapidly gained attention among HF specialists since they were able to show a beneficial prognostic impact in patients affected by HF and T2DM, even independently from the glycemic control as suggested by the EMPA-REG OUTCOME and CANVAS trials.Areas covered: The present review focuses on the mechanisms and the current clinical evidence supporting the use of SGLT2i in HF patients with T2DM. Moreover, the SGLT2i pharmacokinetic and pharmacodynamic properties will be presented in order to better understand the rationale and the design of the ongoing clinical trials investigating directly the effect of this new class of drugs in patients with HF, even independently from T2DM.Expert opinion: SGLT2i are emerging as an effective and safe therapy for the treatment of T2DM and current evidence has unexpectedly demonstrated a robust cardiovascular protection in HF patients with T2DM. Therefore, ongoing clinical trials are investigating directly the effect of this new class of drugs in patients with HF, even independently from T2DM. However, it is methodologically disappointing that the mechanisms underlying the encouraging results in cardiovascular protection of this drug class are still not fully understood. A better understanding of the pharmacokinetic and pharmacodynamic properties of SGLT2i is necessary in order to better determine the effect of this new class of drugs in patients with HF.